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Determination of virus genomes and differentiation of strains and strain variants facilitate the linkage of biological expression to specific genetic units. For effective management of stem pitting disease of citrus tristeza virus (CTV) by cross-protection, an understanding of these links is necessary. The deliberate field application of a biological agent such as a virus first requires a thorough assessment of the long-term impact before it can be applied commercially. Three CTV sources were genetically characterized as different variants of the T68 strain, and their long-term effects on stem pitting and production were investigated. The different CTV sources were inoculated to ‘Star Ruby’ grapefruit trees and evaluated for a number of biological parameters in a field trial in the Limpopo Province of South Africa over a 10-year period. Significant differences were observed in stem pitting severity, impact on tree growth, yield, and the percentage of small fruit produced. These T68 variants were also associated with different stem pitting phenotypes. The variants differed in only 44 nucleotide positions across their genomes, and these minor genetic differences can therefore be used to identify possible genome regions affecting stem pitting.

Two isolates of the T68 genotype of citrus tristeza virus (CTV) were derived from a common source, GFMS12, by single aphid transmission. These isolates, named GFMS12-8 and GFMS12-1.3, induced stem pitting with differing severity in ‘Duncan’ grapefruit (Citrus × paradisi [Macfad.]). Full-genome sequencing of these isolates showed only minor nucleotide sequence differences totaling 45 polymorphisms. Numerous nucleotide changes, in relatively close proximity, were detected in the p33 open reading frame (ORF) and the leader protease domains of ORF1a. This is the first report of full-genome characterization of CTV isolates of a single genotype, derived from the same source, but showing differences in pathogenicity. The results demonstrate the development of intragenotype heterogeneity known to occur with single-stranded RNA viruses. Identification of genetic variability between isolates showing different pathogenicity will enable interrogation of specific genome regions for potential stem pitting determinants.

Soybean dwarf virus (SbDV, genus Luteovirus) is a single-stranded positive-sense RNA virus able to infect several legume species. SbDV was first reported in Japan where it was associated with significant yield losses in soybean (Tamada, 1969). Since then the virus has been detected worldwide. In Europe, the virus has only been reported from Germany (Abraham et al. 2007; Gaafar et al. 2020). In July 2018, several white clover plants (Trifolium repens L.) with leaf discoloration were observed in different locations in Oulu region in northern Finland. Individual plants were collected and analysed for the presence of viruses using small-RNA (sRNA) sequencing (Kreuze et. al. 2009) and reverse transcription-PCR (RT-PCR). Total RNA was extracted using EZNA micro RNA kit (Omega Bio-Tek, GA, USA). For sRNA analysis, sequencing libraries were constructed using the TruSeq small RNA library prep kit (Illumina, CA, USA) and sequenced on Illumina MiSeq platform. On average, 1.3 million single-end reads were obtained per sample, of which 27% were 18-25 nt long and used for the subsequent analysis. Contig assembly and virus identification with VirusDetect software (Zheng et al. 2017) detected SbDV in five out of six white clover samples analysed. Depending on the sample, 26-39 contigs (with lengths up to 301-469 nt) aligned to complete genome of a SbDV isolate previously described from white clover in USA (accession no. JN674402). The cumulative alignment coverage ranged from 35.5 % to 65.3 % with nucleotide identities between 94.4 % and 97.3 %. Additionally, two samples seemed to contain an unidentified closterovirus and one contained White clover cryptic virus 2. No additional viruses were detected from two of the samples.To confirm the presence of SbDV, the samples were tested by RT-PCR using primers MDF, MYF and MUR in multiplex (Schneider et al. 2011) together with SuperScript III One-Step RT-PCR System with the Platinum Taq DNA polymerase kit (Thermo Fisher Scientific, USA), essentially as instructed by the manufacturer. RT-PCR product of approximately 400 bp was produced from each of the five samples previously tested SbDV positive by sRNA analysis. No products were produced from the sample that was SbDV negative in sRNA analysis. Direct sequencing of two of the PCR products produced 347 and 361 bp sequences (GenBank: MZ355392 and MW929169) that were 95.7 % and 95.2 % identical, respectively, to a SbDV isolate (accession no. AB038148) that causes yellowing on soybean and is transmitted by Acyrthosiphon pisum (Terauchi et al. 2003). To our knowledge this is the first report of SbDV in Finland. SbDV is transmitted only by aphids (neither mechanical nor seed transmission occurs). In siRNA analysis all the isolates from Finland formed contigs that aligned almost perfectly (100 % coverage with ≥ 99 % nucleotide identity) to the coat protein (accession no. EF466131) of an SbDV isolate transmittable from white clover to faba bean by A. pisum (Abraham et al. 2007), an aphid common in Finland. Although significant yield losses by SbDV have only been reported on soybean (Tamada, 1969), the virus also causes symptoms in other legume crops, such as growth reduction on pea (Tian et al. 2017) and faba bean (Abraham et al. 2007), both of which are cultivated in Finland. References: Abraham et al. 2007. Plant Dis. 91: 1059. Gaafar et al. 2020. Front microbiol. 11: 583242. Kreuze et al. 2009. Virology 388:1. Schneider et al. 2011. Virology 412: 46. Tamada. 1969. Ann Phytopathol Soc Jpn. 35: 282. Terauchi et al. 2003. Phytopathology 93: 1560. Tian et al. 2017. Viruses 9: 155. Zheng et al. 2017. Virology 500: 130.

The distinguished intracellular stylet puncture called phloem-pd (potential drop [pd]) produced by Myzus persicae has been associated with the transmission of the semipersistently transmitted, phloem-limited Beet yellows virus (BYV, Closterovirus). However, the production of intracellular punctures in phloem cells (phloem-pd) by other aphid species and their role in the transmission of persistently transmitted, phloem-limited viruses are still unknown. Previous studies revealed that inoculation of the persistently transmitted, phloem-limited Barley yellow dwarf virus (BYDV, Luteovirus) is associated mainly with the sieve element continuous salivation phase (E1 waveform). However, the role of brief intracellular punctures that occur before the E1 phase in the inoculation of BYDV by aphids is unknown. We aimed to investigate whether the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae) produced a stereotypical phloem-pd and to study its role in the inoculation of BYDV. The feeding behavior of viruliferous R. padi individuals in barley (Hordeum vulgare) was monitored via the electrical penetration graph (EPG) technique. The feeding process was artificially terminated after the observation of specific EPG waveforms: standard-pds, phloem-pd, and E1. Analysis of the EPG recordings revealed the production of a phloem-pd pattern by R. padi, in addition to a short, distinct E1-like pattern (short-E1), both resulting in successful inoculation of BYDV. Also, the transmission efficiency of BYDV was directly proportional to the time spent by aphids in intracellular salivation in phloem cells. Finally, we discussed the main differences between the inoculation process of semipersistent and persistently transmitted phloem-limited viruses by aphids.

Tristeza, caused by citrus tristeza virus (CTV; Closterovirus, Closteroviridae), is of significant economic importance. Tristeza epidemics have caused severe declines in productivity, and even death, of millions of citrus trees on sour orange rootstock in many regions all over the world. In the field, CTV is most efficiently vectored by the brown citrus aphid (Toxoptera citricida (Kirkaldy)) in a semipersistent manner. The transmission efficiency of the vector is influenced by its acquisition access period (AAP) for CTV. A real-time RT-PCR assay using SYBR Green fluorescent dye was used to estimate the CTV titers in groups of 15 aphids under AAPs after 0.5 to 48 h for three CTV isolates (CT11A, CT16-2, and CTLJ). Similar trends for CTV titer in viruliferous aphids were displayed for the three isolates. The maximum CTV titer was at AAP 6 h for isolates CT11A and CT16-2, and at 4 h for isolate CTLJ. During the AAPs from 0.5 to 6 h, the mean CTV titer of CT16-2 increased from 7.8 × 104 to 1.71 × 107 copies per 15 aphids, and was correlated with an increase in transmission rate from 20 to 90.9%. This suggests that the transmission efficiency is positively correlated with viral titer in the insect from 0.5 h until 6 h AAPs. While a downward trend in CTV titer was observed after a 6-h AAP, the transmission rate remained higher than 90% up to 48 h. These results indicate that factors other than the virus titer in the vector contribute to successful transmission under long acquisition conditions. This is the first detailed quantitative analysis of CTV in its main vector species following different AAPs and its association with transmission efficiency, and should enhance our understanding of T. citricida-CTV interactions.

In the Napa Valley of California, vineyards of ‘Cabernet Franc’ (CF) clone 214, ‘Cabernet Sauvignon’ clone 337, and ‘Zinfandel’ clone 1A (Z1A) with grapevines exhibiting foliar symptoms of red blotches, marginal reddening, and red veins that were accompanied by reduced sugar accumulation in fruit at harvest were initially suspected to be infected with leafroll-associated viruses. However, reverse-transcription polymerase chain reaction (PCR) tests were negative for all known leafroll-associated viruses, with the exception of Grapevine leafroll-associated virus 2 in Z1A. Metagenomic analysis of cDNA libraries obtained from double-stranded RNA enriched nucleic acid (NA) preparations from bark scrapings of dormant canes on an Illumina platform revealed sequences having a distant relationship with members of the family Geminiviridae. Sequencing of products obtained by PCR assays using overlapping primers and rolling circle amplification (RCA) confirmed the presence of a single circular genome of 3,206 nucleotides which was nearly identical to the genome of a recently reported Grapevine cabernet franc-associated virus found in declining grapevines in New York. We propose to call this virus “Grapevine red blotch-associated virus” (GRBaV) to describe its association with grapevine red blotch disease. Primers specific to GRBaV amplified a product of expected size (557 bp) from NA preparations obtained from petioles of several diseased source vines. Chip bud inoculations successfully transmitted GRBaV to test plants of CF, as confirmed by PCR analysis. This is the first report of a DNA virus associated with red blotch disease of grapevines in California.

Citrus tristeza virus (CTV) is a phloem-limited Closterovirus that produces a variety of symptoms in various Citrus spp. One of these symptoms is stem pitting (SP). SP does not occur in all Citrus spp. but when it does it may cause low tree vigor, decline, and an economic reduction in fruit size and yield. Historically, the first appearance of CTV-SP in a citrus area often occurs after the introduction of the most efficient CTV vector, the brown citrus aphid (BCA), Toxoptera citricida. Hypotheses for this association range from the introduction of these strains in new planting materials to the increased ability of BCA to transmit SP strains from existing CTV sources. It is known that CTV often exists as a complex of isolates or subisolates. Single and multiple BCA transmissions have been used to separate different genotypes or strains of CTV from mixed CTV infected plants. This study was initiated to determine what the BCA transmits when an exotic severe SP CTV isolate B12 from Brazil or B408 from Dominican Republic are mixed with a non-SP (NSP) isolate, FS627 from Florida. Biological and molecular data was generated from grafted mixtures of these isolates and their aphid-transmitted subisolates. Single-strand conformation polymorphism patterns of the 5′ terminal region of open reading frame (ORF) 1a, the overlapping region of ORF1b and ORF2, and the major coat protein gene region of NSP and SP CTV-grafted plants remained unchanged but the patterns of doubly inoculated plants varied. The haplotype diversity within SP isolates B12, B408, and mixtures of NSP and SP isolates (FS627/B12 and FS627/B408) and aphid-transmitted subisolates from doubly inoculated plants was determined by analysis of the haplotype nucleotide sequences. Aphid transmission experiments, symptoms, and molecular analyses showed that SP-CTV was more frequently transmitted with or without NSP-CTV from mixed infections.

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

Roy, A., and Brlansky, R. H. 2009. Population dynamics of a Florida Citrus tristeza virus isolate and aphid-transmitted subisolates: Identification of three genotypic groups and recombinants after aphid transmission. Phytopathology 99:1297-1306. Tristeza is an important citrus disease affecting the viability and productivity of citrus worldwide. The causal agent, Citrus tristeza virus (CTV), usually occurs as a mixture of genotypes in nature, with one of the genotypes often dominating the population. CTV has a monopartite, positive-sense RNA genome of ≈19.3 kb and exhibits over 30% diversity in the 5′ half and less than 10% in the 3′ half among different genotypes. A Florida CTV isolate, FS627, was selected for this study. Isolate FS627 was analyzed by reverse-transcription polymerase chain reaction (RTPCR) using primers to three regions: 788-bp region in the 5′ (697 to 1,484 nucleotides), open reading frame (ORF)1a, 696 or 718 bp from the overlapping region of the RdRp (ORF1b) and p33 (ORF2) gene, and a 672-bp major coat protein gene (ORF7) in the 3′ end of the CTV genome. The presence of T36, T30, and VT genotypes in isolate FS627 was confirmed utilizing the genotype specific overlapping region of RdRp primer pairs for RT-PCR amplification followed by cloning and sequence analysis. Analysis of single-strand conformational polymorphisms and sequences of RT-PCR-amplified products of the above regions were used to determine the presence of genotypes in both the parent and aphidtransmitted (AT) subisolates. Although the parent isolate had T36 as the major genotype, T30 was the major genotype in most of the AT subisolates. Some intermediate genotypes were detected that differed from the parental or AT subisolates. These intermediate genotypes were considered to be recombinants of the T30 and T36 genotypes and also were observed in the second level of AT subisolates generated from the of first-level AT subisolates of CTV-FS627. This work provides advance information on the population dynamics in CTV mixtures and the generation of virus recombinants after aphid transmission.

A well-studied severe isolate of Citrus tristeza virus (CTV) known as SY568 has previously been shown to contain multiple variants of the virus which differ in their genetic and biological characters. Aphid transmission was used in an attempt to segregate some of these variants for further characterization. Resulting infections gave symptoms which varied from asymptomatic to more severe than the inoculum source. RNase protection assays (RPAs) were used to compare nine regions of the CTV genome and determine whether unique strains could be identified. Five aphid-transmitted subcultures, with fingerprints that were different from those of the inoculum sources in at least one genomic area, were then cloned, sequenced, and compared with known isolates. An asymptomatic strain was shown to be different in every area of the CTV genome when examined by RPA and sequencing of selected regions. Mixed-infection studies using graft transmission of the asymptomatic subculture and two of the more severe aphid-transmitted subcultures showed that the mild strain was not able to compete well when in the presence of any of the severe variants tested, and its titer was significantly reduced from that seen in single infection. The mild strain and a selected severe strain were singly graft inoculated into five different citrus hosts (sweet orange, grapefruit, sour orange, lemon, and lime), where they maintained their distinct biological and genetic characteristics.

Iracheta-Cardenas, M. M., Metheney, P., Polek, M. L., Manjunath, K. L., Lee, R. F., and RochaPena, M. A. 2009. Serological detection of Citrus tristeza virus with antibodies developed to the recombinant coat protein. Plant Dis. 93:11-16. Antibodies specific for the recombinant coat protein (rCP) of the p25 gene of Citrus tristeza virus (CTV) were developed in goats and rabbits and further evaluated as a complete kit for the detection of the virus using healthy and CTV-infected tissue. The combination of goat T1 used as primary (coating) and rabbit C3 as intermediate (detecting) rCP antibodies reacted efficiently, with optical density at 405 nm (OD405) values between 0.250 and 2.000 with samples from an international collection of diverse CTV isolates. The CTV isolates tested cause a broad spectrum of disease syndromes in different citrus hosts. The OD405 values for healthy tissue were less than 0.100. Likewise, the combination of goat T1 and rabbit C3 rCP antibodies gave consistent results for CTV-positive and -negative sample discrimination when directly compared with the Central California Tristeza Eradication Agency (CCTEA) antibodies used for large-scale CTV detection and a commercially available CTV serological detection kit. The combination of goat T1 and rabbit C3 rCP antibodies showed its suitability for large-scale indexing with samples collected in commercial groves as part of the CCTEA’s regular monitoring program. The evaluation included 41,195 samples from 301 commercial groves from districts 1, 2, and 3. In total, 26 trees (0.063%) were found to be CTV positive using the T1/C3 rCP antibody combination. Results of this research provide evidence that rCP antibodies can be efficiently used for both capturing and detecting CTV antigens in double-antibody sandwich indirect enzyme-linked immunosorbent assay.

Reverse transcription–polymerase chain reaction (RT-PCR) assays were developed for the detection of Citrus tristeza virus (CTV; genus Closterovirus) and Citrus psorosis virus (CPsV; genus Ophiovirus) in citrus trees. Real-time TaqMan RT-PCR was also developed for the detection of CTV. Three different sample preparation methods were compared. The total RNA extraction method by Qiagen was found to be more reliable than the other two methods consisting of crude plant sap extraction and total nucleic acid trapping on a silica bed. Of 287 samples tested for CTV, 210 samples tested positive by RT-PCR and 198 samples by enzyme-linked immunosorbent assay (ELISA). Furthermore, the results from monthly tests of a selected number of field-grown CTV-infected trees showed that RT-PCR detected the virus in 100% of the infected trees in winter and summer, whereas ELISA did not. The one-tube RT-PCR detection was developed for CPsV and was more sensitive than ELISA. Notably, three of 10 CPsV isolates were not detected by ELISA. As demonstrated here, our approach allows the efficacious detection of different viruses in citrus plants using a minimal amount of tissue during all seasons. The molecular methods described could be used in citrus certification programs and to test trees in nurseries and commercial orchards.

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10-3or 10-4extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.

Tzanetakis, I. E., Wintermantel, W. M., Cortez, A. A., Barnes, J. E., Barrett, S. M., Bolda, M. P., and Martin, R. R. 2006. Epidemiology of Strawberry pallidosis-associated virus and occurrence of pallidosis disease in North America. Plant Dis. 90:1343-1346. Strawberry pallidosis-associated virus (SPaV) was found closely associated with pallidosis disease. The modes of transmission of the virus were studied, including pollen, seed (achene), and whitefly transmission. Three whitefly species were tested for their ability to transmit SPaV, but only the greenhouse whitefly, Trialeurodes vaporariorum, was identified as a vector of the virus. Testing strawberries for SPaV and Beet pseudo yellows virus (BPYV), a second crinivirus associated with pallidosis disease, in strawberry-producing areas in North America confirmed a high incidence of both viruses in areas where high populations of whiteflies were present. Infection rates as high as 90% for SPaV and 60% for BPYV were observed when plants exhibiting decline symptoms were tested. Lower rates of infection were found in regions where whiteflies were absent or found in low numbers. The role of these criniviruses in the strawberry decline observed over the past few years along the western coast of North America was examined.

RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3′ untranslated region (UTR); and pCTV2 contained CP, p23, and 3′ UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.

An improved direct tissue blot immunoassay (DTBIA) procedure for detection of Citrus tristeza virus (CTV) within 1 h is described. Prints of fresh young stems of citrus plants that were infected or not infected with CTV were made by gently and evenly pressing the fresh-cut surface of the stems onto a nitrocellulose membrane. The tissue blots were air-dried for 5 min, incubated with prereaction solutions of CTV-specific antibodies and labeled secondary antibodies, goat anti-mouse Ig (H+L)-alkaline phosphatase conjugate or goat anti-rabbit IgG alkaline phos-phatase conjugate, for up to 20 min, rinsed with PBST buffer for 5 min, and immersed into an NBT-BCIP substrate solution for 15 to 20 min. Then the blots were rinsed in water for a few seconds to stop the reactions, and the results were observed and recorded under a light microscope. All samples from greenhouse plants that were infected with CTV decline inducing isolate T-36 were positive to CTV-specific polyclonal antibody 1212 (PCA 1212) and monoclonal antibodies 17G11 (MAb 17G11) and MCA13 (MAb MCA13), whereas samples from greenhouse plants infected with non-decline-inducing isolate T-30 were positive to PCA 1212 and MAb 17G11, but not to MAb MCA13. The noninfected greenhouse plants were negative to all of the antibodies. The improved DTBIA was at least as reliable as other immunological procedures and almost as reliable as polymerase chain reaction for detecting CTV in field trees. The improved DTBIA enables the detection of CTV within 1 h by having a prereaction of CTV-specific antibodies and labeled secondary antibodies in solutions before they are applied to the tissue blots. This DTBIA procedure may be useful in detecting other plant viruses and other pathogens such as bacteria and fungi.

Aguilar, J. M., Abad, J., and Aranda, M. A. 2006. Resistance to Cucurbit yellow stunting disorder virus in cucumber. Plant Dis. 90:583-586. Three hundred accessions of Cucumis sativus, including wild cucumbers, land races, traditional cultivars, and breeding lines, were evaluated under natural-infection conditions in order to identify potential sources of resistance to Cucurbit yellow stunting disorder virus (CYSDV). Although 100% of the susceptible control plants showed typical yellowing symptoms induced by CYSDV, another 24 C. sativus accessions showed partial or total absence of yellowing symptoms. In contrast, when CYSDV inoculation was carried out under controlled conditions, only two (A1 and A2) of these 24 accessions showed resistance to the virus. The nature of the resistance found in A1 and A2 plants was characterized by studying the pattern of virus accumulation and symptom development under controlled infection conditions, and by analyzing the possible nonpreference of Bemisia tabaci for these accessions under free-choice conditions. There was a delay in the establishment of the CYSDV infection in A1 and A2 plants which was evident shortly after inoculation and in apical leaves of the plants at long times after inoculation. Symptom severity was also less for A1 and A2 than for a susceptible control at 8 and 12 weeks postinoculation. Thus, delayed viral infection appeared to be associated with decreased symptom severity in A1 and A2 plants. Our results also showed nonpreference for plants of the A2 accession by B. tabaci, the CYSDV vector.

While characterizing the agents involved in symptomatology of a variegated mint, Mentha × gracilis ‘Variegata’, a nursery plant with atypical symptoms was examined. This plant, unlike ‘Variegata’, did not exhibit yellow vein banding symptoms but instead had distorted and crinkled leaves. Molecular tests for the three viruses found in ‘Variegata’ clones failed to detect any of these viruses in the plant. Double-stranded RNA was extracted and cloned, disclosing the presence of two unknown viruses. One of the viruses was a novel member of the family Closteroviridae. The complete nucleotide sequence of the virus, designated as Mint virus 1, has been obtained. A detection test was developed, and revealed the presence of the virus in several other mint clones and species. Genomic regions from three additional isolates were examined to investigate the genetic diversity of the virus. Genome and phylogenetic analysis placed Mint virus 1 in the genus Closterovirus and transmission studies have identified the mint aphid, Ovatus crataegarius, as a vector for this new member of the genus Closterovirus.

Citrus tristeza virus (CTV) isolates collected from the Lower Rio Grande Valley in south Texas and east Texas were characterized using citrus indicators and molecular methods. The citrus indicators were Mexican lime (Citrus aurantifolia), sour orange (C. aurantium), sweet orange (C. sinensis) grafted to sour orange, Duncan grapefruit (C. × paradisi), and Madam Vinous sweet orange, with some CTV isolates additionally indexed using the Texas commercial grapefruit cvs. Rio Red and Star Ruby, and Marrs and N-33 sweet orange. Severity ratings used 11 biotype groups or cumulative mean relative indices. Molecular characterization was carried out using poly- and monoclonal antibodies, seven strain-specific probes and single-stranded conformational polymorphism, and all were based on the CTV major coat protein or gene. All Texas CTV isolates produced vein clearing symptoms on inoculated Mexican lime plants. Over half of the CTV isolates tested were placed in biotype groups IX and X (causing decline of sweet orange on sour orange, seedling yellows on sour orange and grapefruit seedlings, and stem pitting of grapefruit or sweet orange), and one isolate was in biotype I (mild).

Halbert, S. E., Genc, H., Cevik, B., Brown, L. G., Rosales, I. M., Manjunath, K. L., Pomerinke, M., Davison, D. A., Lee, R. F., and Niblett, C. L. 2004. Distribution and characterization of Citrus tristeza virus in south Florida following establishment of Toxoptera citricida. Plant Dis. 88:935-941. The incidence of Citrus tristeza virus (CTV) was found to increase significantly in southern Florida within 2 years after the establishment of its most efficient vector, Toxoptera citricida (Kirkaldy). Increased incidence of both mild and severe strains was documented, with the incidence of severe strains increasing more than mild strains. Molecular probes capable of differentiating mild, quick decline and various types of stem-pitting strains demonstrated that trees often were infected with more than one strain of CTV, with trees containing up to five different strains. Some CTV strains detected in the southeast urban corridor of Florida and in commercial groves in southwest Florida were found to react with probes specific for stem-pitting strains known from elsewhere in the world. The implications of the presence of these CTV strains in Florida and their possible presence in citrus budwood scion trees are discussed.

The pattern of accumulation of Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, family Closteroviridae) RNA has been analyzed in several cucurbit accessions. In susceptible accessions of melon (Cucumis melo), cucumber (Cucumis sativus), marrow (Cucurbita maxima), and squash (Cucurbita pepo), CYSDV RNA accumulation peaked during the first to second week postinoculation in the first to third leaf above the inoculated one; younger leaves showed very low or undetectable levels of CYSDV. Three melon accessions previously shown to remain asymptomatic after CYSDV inoculation under natural conditions were also assayed for their susceptibility to CYSDV. Hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis of noninoculated leaves showed that only one of these, C-105, remained virus-free for up to 6 weeks after whitefly inoculation. In this accession, very low CYSDV levels were detected by RT-PCR in whitefly-inoculated leaves, and therefore, multiplication or spread of CYSDV in C-105 plants appeared to remain restricted to the inoculated leaves. When C-105 plants were graft inoculated, CYSDV RNA could be detected in phloem tissues, but the systemic colonization of C-105 by CYSDV upon graft inoculation seemed to be seriously impeded. Additionally, in situ hybridization experiments showed that, after C-105 graft inoculation, only a portion of the vascular bundles in petioles and stems were colonized by CYSDV and virus could not be found in leaf veins. RT-PCR experiments using primers to specifically detect negative-sense CYSDV RNA were carried out and showed that CYSDV replication took place in graft-inoculated C-105 scions. Therefore, the resistance mechanism may involve a restriction of the virus movement in the vascular system of the plants and/or prevention of high levels of virus accumulation.

Some strains of the Citrus tristeza virus (CTV) cause stem pitting in sweet orange (Citrus sinensis (L.) Osbeck). This abnormality causes tree decline and reduction in fruit size and yield of affected citrus trees. Stem-pitting symptoms can occur on trunks, on all sizes of limbs, and on the twigs where fruit are produced. Variously sized pits or grooves in the wood often contain a yellow gum. Irregular growth of the phloem occurs in the area of these xylem pits. The histology of stem pitting caused by an Australian CTV isolate was studied in sweet orange using light and electron microscopy. Using scanning electron microscopy, details of the wood pits containing the gumming material were revealed. In thin sections of bark tissue, outgrowths of the phloem tissue were found at various intervals that corresponded to the pits in the wood. Higher numbers of viral inclusions were detected in the phloem outgrowths than were present in the other sieve elements.

Hughes, G., Gottwald, T. R., and Yamamura, K. 2002. Survey methods for assessment of Citrus tristeza virus incidence in urban citrus populations. Plant Dis. 86:367-372. This article concerns survey methodology for pathogens in urban citrus populations, motivated in particular by the need for assessments of Citrus tristeza virus (CTV) incidence. We envisage a large area R not devoted primarily to the commercial cultivation of citrus, that nevertheless has a substantial population of citrus trees. It is desired to sample the citrus population of area R in order to be able to make a statement about the level of infection of the population with CTV, or with particular isolates thereof. We describe a two-stage acceptance sampling scheme in which area R is divided into N sampling units, of which n are inspected. The size of the sam- pling units, while much smaller than R, is still large, so subsampling is carried out, introducing the possibility of misclassification of sampling units. To account for misclassification of sam- pling units, a larger number must be inspected than if it were assumed that there were no misclassifications. We describe the calculation of sample sizes required for subsampling within sampling units and for the total number of sampling units to be inspected, using parameters that can be adjusted to meet different specified regulatory scenarios.

Good, X., and Monis, J. 2001. Partial genome organization, identification of the coat protein gene, and detection of Grapevine leafroll-associated virus-5. Phytopathology 91:274-281. The genome of Grapevine leafroll-associated virus-5 (GLRaV-5) was cloned, and the sequence of 4766 nt was determined. Degenerate oligonucleotide primers designed from the conserved closterovirus heat shock 70 protein (HSP 70) homologue were used to obtain viral-specific sequences to anchor the cloning of the viral RNA with a genomic walking approach. The partial nucleotide (nt) sequence of GLRaV-5 showed the presence of four open reading frames (ORF A through D), potentially coding for the HSP 70 homologue (ORF A); a 51-kDa protein of unknown function with similarity to GLRaV-3 p55 (ORF B); the viral capsid protein (ORF C); and a diverged viral duplicate capsid protein (ORF D). The ORF C was identified as GLRaV-5 viral capsid protein based on sequence analyses and the reactivity of the recombinant protein to GLRaV-5 specific antibodies by western blot analyses. The antiserum produced with the in vitro-expressed GLRaV-5 ORF C protein product specifically reacted with a 36-kDa polypeptide from GLRaV-5 infected vines but did not react with protein extracts from vines infected with other GLRaVs or uninfected vines. Furthermore, specific primers were designed for the sensitive detection of GLRaV-1 and GLRaV-5 by polymerase chain reaction. Grapevine leafroll-associated viruses (GLRaVs) are a group of viruses that collectively or individually cause leafroll disease in grape. To date, at least eight different viruses are associated with the grapevine leafroll disease (24,25). The disease is of economical importance and limits the production of grapes throughout the world (12). Based on virion morphology, GLRaVs are placed in the genus Closterovirus group. Closteroviruses are a large and diverse group of filamentous plant viruses with single-stranded RNA genomes, characteristic genome structure, and generally transmitted by insects (16). The group is classified in the family Closteroviridae, comprising two genera, a monopartite genus Closterovirus with type member Beet yellows virus (BYV), and a bipartite genus Crinivirus with type member Lettuce infectious yellows virus (9,16,20). The viruses associated with grapevine leafroll disease (except GLRaV-2) have coat proteins that appear to be larger in molecular weight than those of the genus Closterovirus. The molecular mass of the characterized GLRaVs coat proteins range between 24 and 43 kDa (5,7,24,27), whereas members of the genus Closterovirus, BYV, Citrus tristeza virus (CTV), and GLRaV-2 have coat proteins with lower molecular mass ranging between 22 and 24 kDa (2,17,19). The partial nucleotide (nt) and deduced amino acid (aa) sequences of the GLRaV-1 (11), the complete GLRaV-2 (1,36) and GLRaV-3 (21) genome nt sequences, and partial GLRaV-4 and GLRaV-5 heat shock 70 protein (HSP) homologue coding sequences (32) have been reported. A comparison of published nt sequences from GLRaV-3 (21) and GLRaV-2 (1,36) indicates that GLRaV-2 is similar to previously characterized closteroviruses (BYV). In contrast, GLRaV-3 is distinct in both genome organization and sequence relatedness. Furthermore, the sequence and molecular data available on genome organization indicates that the order of the capsid protein and diverged duplicate capsid protein is in the reverse order in GLRaV-1 and GLRaV-3, relative to members of the genus Closterovirus (9,16). The viruses associated with leafroll disease are unevenly distributed in grapevine tissues, exist in low concentration, and are frequently found in mixed infections (26). The purification of individual viruses has been difficult, resulting in antisera that cross-react with the different viruses and host proteins, hampering the reliable detection of these viruses in infected plant material (27). We have used molecular methods to overcome the inherent difficulties of serological detection of these viruses. In this paper we report the partial nt sequence of GLRaV-5 genomic RNA. The GLRaV-5 capsid protein was identified and subcloned in a bacterial vector for the overexpression of recombinant protein for the production of a high titer antiserum. Furthermore, polymerase chain reaction (PCR) primers were designed for the sensitive detection of GLRaV-1 and GLRaV-5 RNA in infected grapevines.

Little cherry disease (LChD) is a complex and serious viral disease of cherry. Although originally described almost 70 years ago, there has been little progress in identifying the causal agent of the disease due to the difficulty in obtaining purified virus from infected trees. This problem was partially overcome in 1997 when the compete sequence of a closterovirus associated with LChD, Little cherry virus (LChV), was published. This virus could be associated with some, but not all, trees with LChD, indicating that another virus was also involved. We report here the partial characterization of a second closterovirus associated with LChD, Little cherry virus-2 (LChV-2), and in order to differentiate the two LChD-associated viruses, we refer to LChV as Little cherry virus-1 (LChV-1). LChV-2 is a new closterovirus with molecular similarities to Grapevine leafroll-associated virus-1 (GLRaV-1) and GLRaV-3 but only distantly related to LChV-1. Based on limited sequence comparisons, LChV-2 is the same virus previously identified in association with LChD in Canada. In reverse transcription-polymerase chain reaction detection assays using specific oligonucleotide primers to either LChV-1 or LChV-2, 27 of 28 isolates of LChD tested positive to one or both of these viruses originating from Europe and North America. These results would further confirm the association of LChV-2 with LChD. One isolate, however, tested negative to both LChV-1 and LChV-2, indicating that while this report brings us a step closer to understanding LChD, further work is required to confirm the causal agents of LChD.

Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.

An in situ immunoassay (ISIA) is described for detection of Citrus tristeza virus (CTV). Sections from stems, petioles, or leaf veins of citrus plants that were healthy or infected with CTV were fixed with 70% ethanol and incubated with specific polyclonal antiserum (PCA) 1212 or with monoclonal antibodies (MAbs) MCA13 or 17G11. Bound antibodies were labeled with enzyme-conjugated species-specific secondary antibodies and exposed to a substrate mixture (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate). Presence of CTV antigens was indicated by the development of a purple color, which could be visualized by light microscopy, in the phloem tissues of infected citrus plants. No purple color was observed in the phloem tissues of healthy plants. All isolates used in this study, both severe and mild, were detected by ISIA with the PCA 1212 and the broad spectrum MAb 17G11, but only severe isolates were detected by the strain selective MAb MCA13. Location of CTV antigens could be determined directly and accurately by ISIA in both fresh tissues and samples stored in plastic bags at 4°C or frozen for 4 weeks. Sensitivity of ISIA for detecting CTV in infected plants compared favorably with that of direct tissue blot immunoassay (DTBIA). ISIA is a simple, rapid, specific, and practical procedure for CTV identification applicable to both research and diagnostic needs.

The geographic incidence and molecular variation of three whitefly-borne closteroviruses (lettuce infectious yellows virus [LIYV], cucurbit yellow stunting disorder virus [CYSDV], and beet pseudo-yellows virus [BPYV]) were studied in cucurbits collected from several distinct geographic locations. Of 498 samples analyzed, none were found to be infected by LIYV. Sixty-nine samples collected in the Middle East and Mediterranean Europe were found infected by CYSDV, and twelve samples from Crete and Italy were infected by BPYV. Reverse-transcription poly-merase chain reaction of a portion of the heat shock protein 70 homolog coding region, followed by single-strand conformation polymorphism and nucleotide sequence analysis, was used to estimate the intra- and inter-isolate molecular variability. These analyses showed that each BPYV and CYSDV isolate was composed of a population of sequence variants with a nucleotide identity greater than 98%. CYSDV isolates could be divided into two divergent groups. Group I was only composed of isolates from Spain, Jordan, and Turkey, and group II isolates were predominantly found in Saudi Arabia. Nucleotide identity between isolates of the same group was greater than 99%, whereas identity between both groups was less than 92%. All BPYV isolates showed a nucleotide identity greater than 98%.

Aphid vector species population composition is known to affect the spatial patterns of citrus tristeza virus (CTV) and the changes in these patterns over time. However, the biological processes that are associated with virus spread have not been well defined. The spatiotemporal dynamics of CTV were examined using data collected from research plots in the Dominican Republic and Costa Rica, where the brown citrus aphid (BCA), Toxoptera citricida, was the predominant species, and in Florida, where the BCA was absent and the melon aphid, Aphis gossypii, was the predominant vector. Data were analyzed using a spatiotemporal stochastic model for disease spread, and parameter values were evaluated using Markov chain Monte Carlo stochastic integration methods. Where the melon aphid was the dominant species, the model parameter likelihood values supported the hypothesis that the disease was spread through a combination of random background transmission (transmission originating from inoculum sources outside the plot) and a local interaction (transmission from inoculum sources within the plot) operating over short distances. Conversely, when BCA was present, results often suggested a local short-range transmission interaction that was not restricted to nearest-neighbor interactions and that the presence of background infection was not necessary to explain the observations.

Citrus tristeza virus (CTV) incidence may be assessed by sampling groups of citrus trees, recording the groups as CTV positive (one or more infected trees) or CTV negative (no infected trees), and then calculating disease incidence at the scale of the individual tree by means of a formula involving incidence at the group scale and the number of trees per group. This procedure works well when the CTV status of a tree can be regarded as independent of the CTV status of other trees in the same group. This is the case when the main vector species is Aphis gossypii and the groups comprise four adjacent trees, because the spatial pattern of CTV incidence at the within-group scale can be regarded as random. However, when the main vector species is Toxoptera citricida, this simple procedure is not appropriate, because the spatial pattern of CTV incidence at the within-group scale cannot be regarded as random. Using field data and computer simulation, an alternative procedure for assessment of CTV incidence when the main vector species is T. citricida was devised and tested. In the alternative procedure, the sampling scheme is operationally identical to that used when the main vector species is A. gossypii, but the calculation of CTV incidence at the scale of the individual tree is based on incidence at the group scale and an effective sample size. The analysis of CTV-incidence data collected from a number of citrus blocks in reasonable geographical and temporal proximity and the use of CTV-detection methods more sensitive than the enzyme-linked immunosorbent assay used here are also discussed.

Comparison of a sampling of complementary DNA (cDNA) sequences from the Florida citrus tristeza virus (CTV) isolates T3 and T30 to the sequence of the genome of the Israeli isolate VT showed a relatively consistent or symmetrical distribution of nucleotide sequence identity in both the 5′ and 3′ regions of the 19.2-kb genome. In contrast, comparison of these sequences to the sequence of isolate T36 showed a dramatic decrease in sequence identity in the 5′ proximal 11 kb of the genome. A cDNA probe derived from this region of the T36 genome hybridized to double-stranded RNA (dsRNA) of only 3 of 10 different Florida CTV isolates. In contrast, analogous probes from T3 and T30 hybridized differentially to the seven isolates not selected by the T36 probe. Primers designed from cDNA sequence for polymerase chain reaction (PCR) selectively amplified these 10 isolates, allowing them to be classified as similar to T3, T30, or T36. In contrast, individual cDNA probes derived from the 3′ terminal open reading frames of the T3, T30, and T36 genomes all hybridized to dsRNA from all Florida CTV isolates tested, and PCR primers designed from the T36 capsid protein gene sequence amplified successfully from all isolates. Based on these data, we propose the creation of two groups of CTV, exemplified by the VT and T36 isolates, respectively. Isolates in the VT group, which include isolates VT, T3, and T30, have genomic sequence divergence that is relatively constant in proportion and distribution throughout the genome, and candidate isolates for that group could be considered strains of the same virus. The T36 group is differentiated from the VT group by the highly divergent 5′ genomic sequence. This 5′ region of the CTV genome, thus, can serve as a measure of the extent of sequence divergence and can be used to define new groups and group members in the CTV complex.

Lettuce chlorosis virus (LCV) was purified and partially characterized, and polyclonal antisera were produced and used to assess disease in the field. The antisera reliably detected LCV by indirect enzyme-linked immunosorbent assay (ELISA) in Nicotiana benthamiana. In Western blots, the LCV antisera distinguished between LCV and lettuce infectious yellows virus (LIYV)-infected plants. LCV particle lengths in partially purified preparations, as observed by transmission electron microscopy, were variable, with the majority between 750 and 950 nm long. A single, high molecular weight dsRNA and several lower molecular weight dsRNAs were isolated from LCV-infected N. benthamiana. A single RNA isolated from purified virion preparations was estimated to be 8,625 nucleotides long and was suspected to be the genomic RNA of LCV. LCV was present in experimental field plots in Holtville, California, during the lettuce growing seasons of 1995 to 1997. The percentage of symptomatic plants and yield of lettuce heads treated with insecticide, as well as dsRNA and ELISA reactions for the plots, are reported. A dsRNA consistent in size with LCV was isolated from four weed species in the Imperial Valley of California.

Sether, D. M., Ullman, D. E., and Hu, J. S. 1998. Transmission of pineapple mealybug wilt‐associated virus by two species of mealybug ( Dysmicoccus spp.). Phytopathology 88:1224-1230. Closterovirus-like particles associated with mealybug w ilt of pineapple were acquired and transmitted by the pink pineapple meal ybug, Dysmicoccus brevipes, and the gray pineapple mealybug, D. neobrevipes. Mealybugs acquired pineapple mealybug wilt‐associated virus (PMWaV) from infected pineapple plants or detached leaves. The virus was detected in plants by tissue blot immunoassay and confirmed by immunosorbent electron microscopy. Plants exposed to mealybugs reared on PMWaV-free pineapple tissue remained uninfected. The presence of ants was correlated with an increased rate of virus spread when caged with D. brevipes. All stages of D. neobrevipes acquired PMWaV, although vector efficiency decreased significantly in older adult females. The probability of a single third-instar immature transmitting the virus was 0.04. Both species of mealybug acquired and transmitted PMWaV from infected pineapple material that had been clonally propagated for decades, and both species acquired PMWaV from sources previously infected with the virus by the other mealybug species.

Citrus tristeza virus (CTV) complex comprises a number of isolates or strains producing several economically important disease syndromes in commercial Citrus spp. The stem pitting syndrome is the most important, and causes substantial losses in many citrus-producing regions of the world. In an attempt to develop a serological tool to rapidly differentiate stem pitting isolates of CTV, we evaluated many combinations of trapping and detecting antibodies in an indirect double-antibody sandwich (I-DAS) enzyme-linked immunosorbent assay (ELISA). Two combinations of trapping and detecting antibodies were found suitable for differentiating stem pitting isolates in extracts of infected sweet orange plants. One used a polyclonal serum raised against bacterially expressed CTV coat protein (CP) for trapping and a conformational monoclonal antibody 3E10 for detection, and the other used two polyclonal antisera generated against bacterially expressed CTV CP. Seventy-six CTV isolates from 20 countries, including 35 that cause stem pitting in sweet orange plants, were analyzed in I-DAS-ELISA using different combinations of polyclonal and monoclonal antibodies for trapping and as intermediate detecting antibodies. The ELISA format developed produces a strong positive signal for CTV isolates that cause stem pitting in sweet orange plants and a negative ELISA signal for CTV isolates that do not cause stem pitting. When combined with data on a universal ELISA format, i.e., reacting with a broad range of CTV isolates, these selective ELISA formats allowed reliable serological differentiation of CTV isolates that caused stem pitting in infected sweet orange plants.

Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative 'zinc-finger' domain adjacent to a basic region in p23, the predicted product of ORF II, suggests that this protein might act as a regulatory factor during virus replication., This work was partially supported by grants from the Conselleria de Agricultura y Medio Ambiente, Conselleria de Cultura, Educación y Ciencia (GV-3116/95), INIA (project SC-93111), as well as by contract ERBIC18CT960044 from the European Union

A collection of 27 sources of grapevine rugose wood (RW) disease from a viticultural region in northern Italy was analyzed by graft-inoculating vines of three selective Vitis indicators (V. rupestris cv. St. George, V. berlandieri × V. riparia cv. Kober 5BB, and hybrid cv. LN 33). On the basis of stem reactivity, different groups were identified among the selected RW inoculum sources: nine isolates induced pitting only on cv. St. George, whereas four induced grooving only on cv. Kober 5BB. These two groups were classified as isolates of rupestris stem pitting and Kober stem grooving. Three of the remaining isolates induced wood abnormalities on cvs. LN 33 and Kober 5BB, seven induced wood abnormalities on cvs. St. George and Kober 5BB, and four induced symptoms on all three indicators. These groups may represent RW sources with various disease combinations. RW-affected grapevine clones used as inoculum sources also were tested for virus infections by enzyme-linked immunosorbent assay (ELISA). ELISA revealed the presence of grapevine fleck virus, grapevine leafroll-associated closterovirus 1 and 3, and grapevine trichovirus A. These viruses infected most of the selected RW sources. However, eight of the latter were ELISA-negative. The findings are discussed, and the biological and etiological complexity of the RW phenomena in grapevine is confirmed.

A rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection of citrus tristeza virus (CTV) in three aphid species. Seven CTV isolates from a worldwide isolate collection were used for aphid acquisition feeding by three aphid species. These included the most efficient CTV vector, the brown citrus aphid, Toxoptera citricida; the melon aphid, Aphis gossypii; and the green peach aphid, Myzus persicae, a non-vector for CTV. A short procedure for nucleic acid extraction from single or groups of aphids was developed. Nucleic acid extracts from 1, 3, 5, and 10 aphids with acquisition-access periods of 24 and 48 h were reverse transcribed and amplified using primers for the coat protein gene of the Florida B3 (T-36) isolate of CTV. PCR-amplified fragments of approximately 670 bp were obtained from all the isolates tested and the amplified product from the aphids fed on citrus infected with isolate B3 was confirmed as the CTV coat protein gene by digesting with various restriction enzymes. This technique will be useful in investigations of CTV-vector-plant interactions and CTV epidemiology.

Laboratory studies were conducted on the characteristics of beet yellows closterovirus (BYV) transmission to sugar beets by the bean aphid, Aphis fabae. The relative transmission efficiency and the retention, acquisition, and inoculation thresholds were evaluated using clonal aphid colonies. Transmission efficiency was studied between different apterous aphid species (A. fabae and Myzus persicae ), between alate and apterous morphs of A. fabae and M. persicae, and among five clonal aphid colonies of A. fabae. Results indicated, on average, that apterous M. persicae transmitted twice as efficiently as apterous A. fabae (60 and 34%, respectively), apterous forms of A. fabae were equally efficient in transmitting BYV as compared with alate A. fabae (28 and 29%, respectively), and the five clonal colonies of A. fabae ranged from 28 to 40% transmission efficiency with an average of 34%. The maximum retention time recorded for BYV by A. fabae was between 24 and 48 h. After a 1-h acquisition access, a transmission efficiency of 10% was obtained, with maximum transmission efficiency (the highest average percent transmission that is statistically different from all other previous averages) of 39% reached after 6 h of acquisition access. Inoculation thresholds occurred within 1 h, with maximum transmission efficiency (42%) reached after a 6-h inoculation access. Using an electronic monitoring system, a range of 9 to 38 min (20.5 mean) was recorded for the stylets of A. fabae to reach the phloem sieve elements. This was consistent with our threshold of a 20-min acquisition access recorded through direct observation.

Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.

The effectiveness of five different aphid control regimes in delaying movement of mild and severe isolates of citrus tristeza virus (CTV) into a CTV-free sweet orange scion on sour orange rootstock block was monitored annually for 5 years, using severe isolate-specific and isolate-nonspecific monoclonal antibodies. The average percentage of trees infected with a severe isolate of CTV was 32, 32, 20, 25, and 28 for trees treated with Temik, Temik + Meta-Systox R (MSR), MSR, stylet oil, or no aphid control, respectively, at the conclusion of the experiment (5 years). These percentages were not significantly different (P ≤ 0.05). The average percentage of trees infected only with mild isolates was 13, 10, 7, 7, and 17 for the above treatments, respectively. The infection with mild isolates was significantly greater (P ≤ 0.05) with no aphid control than with stylet oil or MSR treatments.

In April 2009, chlorosis, yellows, and bleaching accompanied with green veins and brittleness on the lower leaves of cantaloupe (Cucumis melo L.) were observed in Lunbei Township, Yunlin County, Taiwan. The same symptoms were also found on cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duchesne), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), bottle gourd (Lagenaria siceraria (Molina) Standl.), and oriental pickling melon planted in other areas of Yunlin and Changhua counties in central Taiwan. Large populations of whiteflies were observed in association with the diseased cucurbit crops, and they were further identified as silverleaf whitefly (Bemisia argentifolii Bellows & Perring) by PCR with specific primers BaBF (5′-CCACTATAATTATTGCTGTTCCCACA-3′) and l2-N-3014R (5′-TCCAATGCACTAATCTGCCATATTA-3′) (3). In June 2009, samples from symptomatic cantaloupe were collected for virus diagnosis. Flexuous filamentous virions of 700 to 900 nm were observed in crude sap of the symptomatic cantaloupe tissues with transmission electron microscopy. On the basis of the suspected insect vector, symptomology, and virus morphology, a Crinivirus species was suspected as the causal agent. A nested reverse transcription (RT)-PCR assay with degenerate deoxyinosine-containing primers developed for detection of Closterovirus and Crinivirus (1) was conducted. Total RNAs extracted from 16 symptomatic cantaloupe samples with a Plant Total RNA Miniprep Purification Kit (Hopegen, Taichung, Taiwan) were analyzed, and a 0.5-kb DNA fragment was amplified from eight of them. The PCR products were sequenced and the sequences were identical among samples. A comparison of the submitted sequence (Accession No. HM120250) with those in GenBank showed that the sequence was identical to the Hsp70h sequences of Cucurbit chlorotic yellows virus (CCYV) isolates from Japan (Accession No. AB523789) (4) and China (Accession Nos. GU721105, GU721108, and GU721110). To identify CCYV infection in the field, the specific primers, Crini-hsp70-f (5′-GCCATAACCATTACGGGAGA-3′) and Crini-hsp70-r (5′-CGCAGTGAAAAACCCAAACT-3′), that amplify a 389-bp DNA fragment corresponding to the nucleotide 1,324 to 1,712 of RNA2 of the original CCYV Japan isolate (Accession No. AB523789) were designed for detection of CCYV. In RT-PCR analyses, CCYV was identified in cantaloupe (305 of 599 samples), watermelon (27 of 93 samples), cucumber (all 15 samples), melon (82 of 92 samples), pumpkin (8 of 10 samples), and bottle gourd (10 of 17 samples) showing chlorosis and yellowing. The 389-bp DNA fragment was also amplified by RT-PCR with the primer pair Crini-hsp70-f/Crini-hsp70-r from total RNA extracts of 29 of 116 silverleaf whitefly individuals collected from the diseased cantaloupe fields in Lunbei Township from August to October, 2009. CCYV is a newly characterized Crinivirus species, first discovered in Japan in 2004 (2) and also found in China in 2009. To our knowledge, this is the first report that CCYV is emerging as a threat to cucurbit productions in Taiwan. References: (1) C. I. Dovas and N. I. Katis. J. Virol. Methods 109:217, 2003. (2) Y. Gyoutoku et al. Jpn. J. Phytopathol. 75:109, 2009. (3) C. C. Ko et al. J. Appl. Entomol. 131:542, 2007. (4) M. Okuda et al. Phytopathology 100:560, 2010.

A Kenyan isolate of sweet potato sunken vein virus (SPSVV-Ke), a tentative member of the genus Closterovirus, was transmitted to Ipomoea setosa by the whitefly Bemisia tabaci. Cross-banded filamentous particles about 850 nm in length were detected in infected plants by immunoelectron microscopy (IEM) with an antiserum to virions of an Israeli isolate of SPSVV (SPSVV-Is). Viral double-stranded RNA species of about 10 and 9 kbp were extracted from infected I. setosa and used as templates for complementary DNA (cDNA) synthesis. Sequencing of selected cDNA clones revealed an open reading frame of 774 nucleotides that encodes a protein with an estimated molecular mass of 29,028 Da. Computer analysis of the deduced amino acid sequence of this protein indicated a distinct affinity to the coat protein (CP) of lettuce infectious yellows closterovirus (LIYV) and a lesser similarity to the CPs of beet yellows and citrus tristeza closteroviruses, suggesting that it is the CP of SPSVV-Ke. After expression of the CP gene of SPSVV-Ke in Escherichia coli, its identity as the viral CP was confirmed by Western blot analysis with the SPSVV-Is antiserum. This antiserum and a rabbit antiserum raised against the bacterially expressed CP of SPSVV-Ke were used in Western blot and IEM experiments for assessing the serological relationships among SPSVV-Ke, SPSVV-Is, and sweet potato virus disease-associated closterovirus isolates from Nigeria and the United States. Results showed that SPSVV-Ke is closely related serologically to similar closterovirus isolates infecting sweet potato in Israel, Nigeria, and the United States but differs from them in reacting weakly with an antiserum to LIYV in IEM and Western blots.

A national survey was conducted in Canada during 1994 and 1995 for four viral pathogens of quarantine significance in grapevine. A total of 11,417 samples, collected from 637 field sites, was tested for the presence of arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), and two viruses commonly associated with grapevine leafroll disease, grapevine leafroll associated virus types I (GLRaV-I) and III (GLRaV-III). Nationally, the incidence of nepovirus-infected samples was low, 0.53 and 0.25% for ArMV and GFLV, respectively, and higher for the two leafroll associated viruses, 1.67% for GLRaV-I and 10.8% for GLRaV-III. Nepoviruses were found only in samples of varieties of Vitis vinifera origin or hybrid crosses, whereas GLRaV-I and GLRaV-III were found in samples of all variety types. Among the varieties tested, GLRaV-III was found primarily in samples of hybrid (14.8%) or other (including the Labrusca varieties Concord, Niagara, and Elvira) (13.5%) origin, whereas GLRaV-I was found predominantly in samples of V. vinifera (4.05%) origin. The widespread occurrence of these viruses in Canada has resulted in a reevaluation of their current quarantine status.

Tomato infectious chlorosis virus (TICV) is a newly described closterovirus. Virions purified from TICV-infected plants contained two single-stranded (ss) RNAs, one of approximately 7,800 (RNA 1) and the other 7,400 (RNA 2) nucleotides. Double-stranded (ds) RNA analysis showed two prominent dsRNAs of approximately 7,800 and 7,400 bp, as well as several smaller dsRNAs. The TICV virion ssRNAs were used for cDNA cloning. Of 200 cDNA clones analyzed, 10 clones containing cDNAs ranging in size from about 900 to 1,500 nucleotides were used to generate digoxigenin-UTP-labeled transcripts. These transcripts hybridized with the TICV ssRNAs in Northern blot hybridization analyses and were used in dot-blot analyses to confirm TICV infection in several host plants including tomato, potato, Physalis wrightii, Nicotiana clevelandii, and artichoke. None of the probes reacted with any uninfected host plant tested or with plants infected with four other clostero- or clostero-like viruses including lettuce infectious yellows closterovirus, lettuce chlorosis virus, cucurbit yellow stunting disorder virus, and beet pseudo yellows virus. Northern blot hybridization analyses using selected riboprobes showed no detectable homology between TICV dsRNA 1 and 2, or between subsets of smaller dsRNAs. Inclusion bodies, characteristic of the closteroviruses, were consistently associated with the phloem of TICV-infected N. clevelandii.

Citrus tristeza virus in Australian citrus is a complex of isolates that differ in biological properties and rates of aphid transmission. Biological characterization of isolates on citrus indicators produced a range of symptoms varying in type and severity depending on the field host and geographical location. Single aphid transmissions with the vector Toxoptera citricida separated some of these subisolates based on biological indexing on three citrus indicators and the numbers of inclusion bodies produced. Inclusion numbers produced in Citrus aurantiifolia were positively related to isolate and subisolate severity in that host.

The release of new virus-free propagation material and new cultivars of grapevine would be advanced if more rapid diagnostic methods could be developed. A method enabling rapid in vitro indexing of grapevine leafroll is described. Diseased explants poorly express symptoms when placed in culture. However, when placed under mild stress conditions (4% sorbitol), a variety of distinct symptoms appear within 4 to 8 weeks. This method could potentially replace the conventional 18- to 30-month indexing procedure.

Oligonucleotide primers were designed based on nucleotide sequences corresponding to the conserved phosphate I and 2 motifs contained within the closterovirus-encoded heat shock protein 70 (HSP70) homolog. These primers were used with RNAs extracted from virus-infected plants and reverse-transcriptase polymerase chain reaction to generate specific cDNAs for lettuce infectious yellows closterovirus (LIYV) and for four additional whitefly-transmitted viruses for which corresponding nucleotide sequence data were unavailable: tomato infectious chlorosis virus (TICV), cucurbit yellow stunting disorder virus (CYSDV), beet pseudo-yellows virus (BPYV), and lettuce chlorosis virus. The resulting cDNAs of approximately 600 bp, corresponding to LIYV, TICV, CYSDV, and BPYV, were cloned, and the nucleotide sequences were determined. Computer-assisted analysis of the deduced amino acid sequences showed that all exhibited significant similarity to the HSP70 proteins in general and to the HSP70 homologs encoded by closteroviruses in particular. Comparative alignments showed the amino acid sequences for LIYV, TICV, BPYV, and CYSDV were more similar to each other than to the corresponding regions for the HSP70 homologs of three aphid-transmitted closteroviruses. Digoxigenin-11-UTP-labeled transcripts were generated from each cloned cDNA and used in RNA and dot blot hybridization analyses. Probes for LIYV, TICV, BPYV, and CYSDV hybridized only with double-stranded RNAs or extracts of plants infected with the corresponding virus.

The study of citrus tristeza closterovirus (CTV) has been hindered by particle fragility, the inherently low virus titers, and its woody citrus hosts. We report the development of an in vitro Citrus sinensis cv. Hamlin protoplast system useful for the study of CTV replication. Full-length CTV genomic RNA from purified virions was inoculated into 'Hamlin' protoplasts in the presence of polyethylene glycol. After 24 h, the protoplasts were assayed for virus replication. Positive- and negative-strand CTV genomic RNA were detected by Northern hybridization using digoxigenin-labeled RNA probes. Three viral-encoded proteins of 20, 25 (capsid protein), and 27 kDa were detected by Western blotting, demonstrating that the viral RNA was translated in vivo. CTV virus particles also were isolated and observed in the electron microscope. This was the first demonstration of the infectivity of CTV RNA and the first report of CTV replication in protoplasts. This system will enable a more detailed study of the replication strategy of this economically important and complex virus.